Study On Cell Imaging And Antibacterial Potential Of 2’-Fluorodeoxy Uridine Based Fluorescent Probe Via AIE Mechanism

In 2011 Tang Benzhong,a Chinese scientist,discovered an unusual phenomenon based on his research:the more molecules gathered,the greater the fluorescence,which he named as aggregation-induced emission（AIE）.Due to their superior optical characteristics and ability to eliminate conventional aggregation-caused quenching（ACQ）,AIE-based organic fluorescence probes have displayed broad application in chemical sensors,biological probes,and medical diagnosis and therapy.However,its hydrophobic chromophores are composed of many aromatic rings and bulky steric structure with branches,define its properties,such as limited water solubility and biological compatibility,which restrict its applicability in the biomedical area.How to construct multi-functional AIE fluorescence probe with high biocompatibility and sensitivity is now a significance research field.In this study,a novel biocompatible nucleoside conjugated tetraphenyl vinyl（TPE）based fluorescence probe via AIE mechanism was developed.Sonogoshira coupling was used to introduced non-natural nucleosides with good biocompatibility into the TPE moiety with AIE effect to improve its biocompatibility and expand its potential application.There are now several studies and reports available on the industrial application of fluorine-containing antibacterial compounds.Fluorine atoms have significant electronegativity and lipophilicity,hence incorporating fluorine atoms into compounds can result in outstanding qualities such as structural stability,lipophilicity,and antibacterial capabilities.In this study,the pyrimidine ring of the non-natural nucleoside（5-iodine-2’-fluoro-deoxyuridine）that have been chemically modified was coupled with the pre-synthesized alkyne functionalized TPE through coupling reaction.A novel nucleoside fluorescent probe based on AIE mechanism was obtained and named TPE-FdU.Methods:1.Chemical synthesis and characterization of TPE-FdU（1）The chemical synthesis of the TPE-FdU:The obtained crude product of TPE-FdU was then purified using High Performance Liquid Chromatography（HPLC）.（2）Identification of the TPE-FdU:The target product was fully identified using various spectroscopic techniques such ¹H,¹⁹F,¹³C nuclear magnetic resonance（NMR）spectrum and mass spectrum.（3）Characterization of the TPE-FdU:The optical potential of compound was evaluated using UV-visible absorption spectra and fluorescence spectroscopy.The AIE characteristic of synthesized TPE-FdU was confirmed by fluorescence spectrum.2.Study of the cell imaging effect of the TPE-FdU fluorescence probe（1）Cytotoxicity of TPE-FdU:The biosafety of TPE-FdU was assessed by MTT as a fluorescent probe.（2）Cell imaging effects of TPE-FdU:The cell imaging potential of TPE-FdU by confocal laser scanning microscopy were evaluated.3.Study of the antibacterial effect of the TPE-FdU fluorescent probeWe also evaluated the antibacterial effect of TPE-FDU by measuring inhibition zone,MIC,MDC and growth curve when TPE-FdU co-cultures with four common gram-positive and gram-negative bacteria.Results:1.Chemical synthesis and characterization of TPE-FdU（1）The Chemical Synthesis and Identification of TPE-FdU:The obtained crude product of TPE-FdU was then purified using HPLC,and the peak that appeared at 11.677 min was collected and concentrated under reduced pressure.The product was obtained as a white solid.The date of¹H-NMR and ¹³C-NMR prove that we have successfully synthesized the compound TPE-FdU.（2）Characterization of the TPE-FdU:The interaction of TPE-FdU with various DNA and RNA was explored using UV-visible and fluorescence monitoring.The fluorescence intensity of the probe was positively correlated with the concentration of RNA,but DNA do not show the same trend under similar experimental conditions,indicating that the TPE-FdU fluorescent probe had a certain specificity for RNA.2.Study of the cell imaging effect of the TPE-FdU fluorescence probe（1）Cytotoxicity of TPE-FdU:Following MTT that the TPE-FdU fluorescence probe had no harmful impact on cells at the effective fluorescence concentration.（2）Cell imaging effects of TPE-FdU:The findings of the confocal laser assay revealed that the TPE-FdU was applied to the cell staining in the cytoplasm rather than the nucleus,which is consistent with the fluorescence spectrum results（the compounds only specificity combined with RNA）.Further,we found that TPE moiety has a great capacity to transmembrane,allowing it to demonstrate cell imaging occurred in a relatively short time.The result showed TPE-FdU had a specific targeting impact on mitochondria based on co-localization assay.3.Study of the antibacterial effect of the TPE-FdU fluorescent probeThe results of the inhibition zone showed that TPE-FdU had a certain antibacterial effect on Staphylococcus aureus,Staphylococcus surface,Escherichia coli and Pseudomonas aeruginosa.The MIC of TPE-FdU against Staphylococcus aureus and Escherichia coli was 0.5 mg/m L and the MBC was greater than 1 mg/m L.The MIC and MBC of TPE-FdU against Staphylococcus surface was 0.5 mg/m L.The MIC of TPE-FdU against Pseudomonas aeruginosa was 0.25 mg/m L and the MBC was greater than 1mg/m L.SH had the strongest inhibitory effect on the growth curve of the four bacteria where the concentration was 4 MIC.Conclusion:All the obtained data show that we effectively developed and manufactured an AIE probe with high stability,specificity,fluorescence,and cell imaging capability,as well as a bifunctional chemical with antibacterial capabilities.TPE-Fdu based fluorescent probe is highly specific for RNA and can be effectively used for the cell imaging of mitochondria.